Some insects compete with us for food and transmit human and livestock diseases, but other insects are beneficial. Therefore, there has been a continuous demand for the development of insect control methods that are target-specific. Juvenile hormone (JH) and ecdysteroids are the major hormones that regulate molting, metamorphosis and reproduction in insects. Since these hormones are not present in vertebrates, they represent attractive targets for the development of insect control methods. Hindering this effort is the lack of understanding of the molecular basis of JH action. The main objective of this proposal is to identify genes that play critical roles in JH action. JH response elements (JHRE) identified in the promoter region of JH- and 20-hydroxyecdysone (20E)-responsive JH esterase (jhe) gene and ecdysone receptor (EcR) deficient Drosophila melanogaster cell line (L57) were used to develop a robust system that can be used for studying the molecular basis of JH action. In L57 cells, a reporter gene placed under the control of JHRE is induced by JH, and the JH induction was suppressed by 20E. The nuclear proteins isolated from L57 cells bind to JHRE and 20E in the presence of EcR can prevent this binding. In L57 cells, EcR and DSP do not bind directly to JHRE and are not directly involved in JH induction of a reporter gene regulated by JHRE. Identification of proteins that bind to JHRE is critical for understanding JH action. The two specific aims of the proposal are: (1) to identify genes that play key roles in JH action; and (2) to determine the role of identified genes in JH action. The genes that are involved in JH induction of JHRE regulated reporter gene will be identified using RNAi and yeast assays. The identified gene will be characterized by studying the binding of expressed proteins to JHRE and labeled JH, expression of their mRNA and proteins during D. melanogaster development, and analysis of D. melanogaster mutants deficient in identified genes. JH analogs are being used for mosquito control, but some mosquitoes are already developing resistance to these analogs. The results from these studies will aid in understanding the mode of action of JH analogs, thus facilitating their judicious use. The genes identified will be useful to develop screening assays to identify new insect disease vector control agents. The receptors can also be used in medicine by developing gene switches for various applications such as gene therapy, drug discovery and therapeutic protein production.